MXPA99004045A - Method for improved selectivity in photo-activation and detection of molecular diagnostic agents - Google Patents

Abstract

A method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photo-active molecular agent. The method comprises the steps of treating the particular volume of the plant or animal tissue with light sufficient to promote a simultaneous two-photon excitation of the photo-active molecular agent contained in the particular volume of the plant or animal tissue, photo-activating at least one of the photo-active molecular agents in the particular volume of the plant or animal tissue, thereby producing at leastone photo-activated molecular agent, wherein the at least one photo-activated molecular agent emits energy, detecting the energy emitted by the at least one photo-activated molecular agent, and producing a detected energy signal which is characteristic of the particular volume of plant or animal tissue. The present invention also provides a method for the imaging of a particular volume of material, wherein the material contains at least one photo-active molecular agent.

Description

METHOD FOR ENHANCED SELECTIVITY IN PHOTOACTIVATION AND DETECTION OF MOLECULAR DIAGNOSTIC AGENTS DESCRIPTION Background and field of the invention The present invention relates in general to methods and apparatus for remotely performing spatially selective photoactivation of one or more molecular agents and for improving the detection of the diagnostic signals produced therewith. The method described for performing photoactivation uses the special properties of nonlinear optical excitation to promote an agent from one molecular energy level to another with a high degree of molecular and spatial specificity. The special features of this method are applicable for activation of several endogenous and exogenous imaging agents, and in particular it provides different advantages in the diagnosis of diseases in humans and animals. Specifically, the use of non-linear excitation methods facilitates the controlled activation of diagnostic agents in deep tissue using near-infrared to infrared radiation, which is absorbed and diffused to a lesser extent than the methods and radiation currently used. The combination of these non-linear excitation methods with advanced signal processing and coding methods greatly increases sensitivity in the detection of diagnostic signals. There is an urgent need in many fields, and especially in the field of medical diagnostics, for a method that is capable of selectively controlling the long-range activation of several molecular agents while producing few, if any, side effects of the process of activation. The desired improvements in activation include improvements in spatial or temporal control over the location and depth of activation and reduction in the undesirable activation of other molecular agents or co-localized or proximal structures, and greater preference in the activation of desirable molecular agents over that of undesirable molecular agents. Several linear and nonlinear optical methods have been developed to provide some of such improvements for some of such agents under very special conditions. However, in general the functioning and applicability of these methods have been lower than desired. Specifically, improved photoactivation methods that can be used to selectively photoactivate a variety of molecular diagnostic agents while providing improved performance in controlling the application of this photoactivation are needed. The application of optical radiation as a means to activate long-distance molecular specimens has been known for many years. Specifically, linear optical excitation methods have been widely used as a means to achieve semi-selective activation of molecular diagnostic agents. Linear optical excitation occurs when an objective agent such as a molecular diagnostic agent undergoes a specific photochemical or photophysical process, such as fluorescent emission, during the absorption of energy provided by a single photon. These processes can in many cases be very efficient, and the use of such processes is attractive for many applications. Unfortunately, the operation of these linear methods has not always had the desired success. For example, there is strong evidence that the ultraviolet radiation used to excite some molecular specimens can cause disease in humans and animals, such as an induced skin cancer, along with other undesirable side effects. In addition, less than desired penetration has clouded most linear optical excitation efforts of molecular therapeutic agents, primarily as a consequence of the optical diffusion and absorbance effects of the incident radiation test at wavelengths close to the bands of linear absorption of these agents. As an example, Watcher and Fisher (EA atcher and WG Fisher, ^ Methods and Aparatus for Evaluating Structural eakness in Polymer Matrix Composites, "United States Patent No. 5,483,338) describe a rapid optical method capable of chemical transformations forming images in sensitive form in molecular testing agents; however, due to the diffusion and absorbance of the incident test radiation, the method is only applicable to topical analysis. Vo-Dinh and his collaborators (T. Vo-Dinh, M. Panjehpour, BF Overholt, C. Farris, FP Buckley III and R. Sneed, X In-Vivo Cancer Diagnosis of the Esophagus Using Differential Normalized Fluorescence (Dnf) Indexes, "Lasers in Surgery and Medicine, 16 (1995) 41-47; and M. Panjehpour, BF Overholt, JL Schmidhammer, C. Farris, PF Buckley and T. Vo-Dinh, ^ Spectroscopic Diagnosis of Esophageal Cancer: New Classification Model, Improved Measurement System, "Gastrointestinal Endoscopy, 41 (1995) 577-581) describes the use of similar optical and linear test methods for detection of diseased tissue in humans; however, this attempt is also clouded by penetration depth less than desired and is limited to the detection of superficial lesions due to diffusion and absorption of the incident test radiation. Also, because this type of excitation is linearly related to the excitation power, these methods do not provide effective means to limit the location of the test excitation along the optical path. In fact, virtually all examples of the use of linear optical excitation are clouded by fundamental performance limits that are attributable to the undesirable diffusion and absorption of optical radiation incident by the surrounding matrix, poor specificity in the excitation of molecular test species. and a lack of adequate physical mechanisms for precise control of the extent and depth of activation. Several nonlinear optical excitation methods have been used in an effort to achieve specific improvements in photoactivation selectivity for certain applications, and to orient many of the limitations of linear excitation methods. In fact, the nonlinear process consists of the simultaneous absorption of two photons of light by a molecule to effect excitation equivalent to that resulting from the absorption of a single photon having twice the energy of these two photons, as well as what are the specific advantages of this process in terms of reduced absorption and diffusion of excitation photons by the matrix, improved spatial control over the excitation region and reduced photophysical and photochemical damage potential for the sample. Excitation sources have been employed ranging from simple continuous wave (CW) lasers to Q-switched pulsatile lasers having peak powers greater than 1 GW for a variety of examples of two-photon excitation methods. For example, Wirth and Lytle (MJ Wirth and FE Lytle, ^ Two-Photon Excited Molecular Fluorescence in Optically Dense Media '*, Analytical Chemistry, 49 (1977) 2054-2057) describe the use of nonlinear optical excitation as a means to stimulate target molecules present in optically dense media; this method proves useful in limiting undesirable direct interaction of the test radiation with the medium itself, and provides a means to effectively excite target molecular agents present in strongly absorbing or diffusing matrices. Further spatial control has been further developed by With (MJ Wirth and HO Fatnumbi, XVery High Detectability in Two-Photon Spectroscopy, "Analytical Chemistry, 62 (1990) 973-976), specifically, Wirth describes a method for achieving extremely high spatial selectivity. high in the excitation of target molecular agents using a microscopic imaging system .. Similar controls have been applied in addition by Denk et al. (W. Denk, JP Strickler and WW Webb, 'Two-Photon Laser Microscopy,' patent of the United States of America No. 5,034,613) who describes a special epi-illumination confocal laser scanning microscope that uses non-linear excitation to achieve intrinsically high three-dimensional spatial control in the photoactivation of several molecular fluorophore agents at a cellular or subcellular scale. DenkHowever, it is specifically oriented to microscopy, where a microscope is used to observe samples on a slide. This microscope is used to excite molecular fluorophore agents added to biological samples or specimens, which constitute an optically dense medium. In Denk, the light coming from a laser travels downwards as a result of being reflected by means of a mirror on a flat object. The fluorescent light then travels back along the same optical path, through a lens, a series of mirrors and finally a photomultiplier tube. Therefore, light only focuses on the flat object of a slide and is reflected back. The special properties of two-photon non-linear optical excitation are used to virtually limit the excitation and subsequent detection of the fluorescent signal produced in this way to a confocal region occurring at the focus of a target lens, with which the contrast in the formation of three-dimensional images through precise control of the depth of focus. The fluorescent light emitted is collected by means of the excitation lens using an epi-lighting configuration. The control of photoexcitation for the generation of luminescence-based images at the cellular and subcellular level is shown in objective samples mounted on a stage. In addition, Denk describes that the reduction in photoinduced necrosis of cells located in the focal plane is a main benefit of this approach or proposal of microscopy, based on the replacement of ultraviolet excitation radiation with the less harmful radiation of excitation near the infrared. . In a later work done by Denk et al. (W. Denk, DW Piston and WW Webb, "Two-Photon Molecular Excitation in Laser Microscopy," in Handbook of Biological Confocal Microscopy, Second Edition, JB Pawley, ed., Plenum Press, New York, 1995, pages 445-458 ) describes an external total area detection method for collection of microscopic imaging data produced from excited two-photon fluorescent labels. This method, which the authors state remains "yet to be experienced," eliminates the need to collect backscattered fluorescent light using epi-illumination (see page 452). Denk points out that this approach could be useful if the objective of the microscope did not transmit the fluorescent wavelengths emitted, but that it is "vulnerable to light pollution from the environment." In this work and in the first Denk patent (5,034,613), no obvious method is used or anticipated for the reduction of background light interference from ambient light or diffused excitation light. In fact, the well-known low efficiency of the two-photon excitation process can result in a very high proportion of diffusion, unabsorbed excitation light for fluorescence emission. The use of various modulation methods for the reduction of interference from diffused excitation light, as well as from interference from ambient light and from other environmental and instrumental background sources, has numerous precedents. In the field of excited two-photon fluorescence, Lytle et al (R.G. Freeman, D.L. Gilliland and F.E. Lytle, "Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence," Analytical Chemistry, 62 (1990) 2216-2219; and W.G. Fisher and F.E. Lytle, "Second Harmonic Detection of Spatially Filtered Two-Photon Excited Fluorescence," Analytical Chemistry, 65 (1993) 631-635) describe sophisticated methods for the rejection of diffused excitation laser light by using second harmonic detection methods: when the sinusoidal modulation of the excitation light is carried out at a frequency and the detection of the excited fluorescence of two photons is performed at twice that frequency (which is the second harmonic of the excitation modulation frequency), the interference coming from light of widespread excitement are virtually eliminated. And by a suitable selection of the modulation frequency to avoid electronic noise and other noise frequencies, the rejection of instrumental and environmental interference is extremely high. Thus, it is well known that the excitation of two fluorescence photons can be used under laboratory conditions to excite molecular fluorophores using light at approximately twice the wavelength of that used for the linear excitation of a single photon, and that the excitation performed in this way can improve three-dimensional spatial control over the excitation location, can reduce interference from absorption and diffusion of excitation light in optically dense media, and can reduce collateral damage along the excitation path for samples of living cells under microscopic examination. However, while the main part of the prior art clearly exemplified by these cited examples shows many attractive features of various photoactivation methods that are applicable for diagnosis and other uses of live microscopic imaging, it has not been shown previously. a general method to achieve selective photoactivation of one or more molecular agents with a high degree of spatial control that is capable of meeting the diverse needs of the medical diagnostic industry. Specifically, practical methods for effecting such control at scales that are significant for medical diagnostic applications have not been described. Accordingly, an object of the present invention is to provide a general method for achieving selective photoactivation of one or more molecular agents with a high degree of spatial control. It is another object of the present invention to provide such a method that is capable of meeting the diverse needs of the medical diagnostic industry.

It is another object of the present invention to provide a practical method for effecting such control at important scales for medical diagnostic applications.

Having considered the above and other objects and advantages, the present invention generally provides a method for the imaging of a particular volume of plant or animal tissue, wherein the plant or animal tissue contains at least one photoactive molecular agent. The method comprises the steps of treating the particular volume of the plant or animal tissue with sufficient light to promote a simultaneous excitation of two photons of the photoactive molecular agent contained in the particular volume of the plant or animal tissue, to photoactivate at least one of the at least one a photoactive molecular agent in the particular volume of the vegetable or animal tissue, thereby producing at least one photoactivated molecular agent, wherein the at least one photoactivated molecular agent emits energy, detecting the energy emitted by the at least one agent photoactivated molecular and produce a detected energy signal that is characteristic of the particular volume of plant or animal tissue. The present invention also provides a method for imaging a particular volume of material, wherein the material contains at least one photoactive molecular agent. In a preferred embodiment of the present invention, sufficient light to promote simultaneous excitation of two photons of the at least one photoactive molecular agent is laser light. It is also preferred that sufficient light to promote a simultaneous excitation of two photons of the photoactive molecular agent is a focused light beam, and more preferred that the focused light beam be focused laser light. Another preferred embodiment of the present invention further includes a first step of treating the material, plant tissue or animal tissue with at least one photoactive molecular agent, wherein the particular volume of the material, plant tissue or animal tissue retains at least a part of at least one photoactive molecular agent. It is more preferred that the at least one photoactive molecular agent is selected from the group consisting of psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 4,5 ', 8-trimethylpsoralen (TMP). , 4 '-aminoethyl-4, 5T, 8-trimethylpsoralen (AMT), 5-chloromethyl-8-methoxypsoralen (HMT), angelicin (isopsoralen), 5-methylangelicine (5-MIP), 3-carboxypsoralen, porphyrin, derivative of hematoporphyrin (HPD), Photofrin II, benzoporphyrin derivative (BPD), protoporphyrin IX (PpIX), hematoporphyrin ether dye (DHE), polyhematoporphyrin (PHE) esters, 13,17-N, N, N-dimethylethyl ethanolamine ester of protoporphyrin (PH1008), tetra (3-hydroxyphenyl) -porphyrin (3-THPP), tetraphenylporphyrin monosulfonate (TPPS1), tetraphenylporphyrin disulfonate (TPPS2a), dihematoporphyrin ether, mesotetraphenylporphyrin, mesotetra (4N-methylpyridyl) porphyrin (T4MpyP) , octa- (4-tert-butylphenyl) tetrapyrazineporphirazine (OPTP), phthalocyanine, tetra- (4-tert-butyl) phthalocyanine ( t4-PcH2), tetra- (4-tert-t-butyl) phthalocyanatomagnesium (t4PcMg), sulfonated chloraluminium phthalocyanine (CASPc), chloraluminium phthalocyanine tetrasulfate (AIPcTS), mono-sulphonated aluminum phthalocyanine (AISPc), phthalocyanine di-sulfonated aluminum (AlS2Pc), tri-sulfonated aluminum phthalocyanine (AlS3Pc), tetra-sulfonated aluminum phthalocyanine (AlS4Pc), silicon phthalocyanine (SiPc IV), zinc phthalocyanine II (ZnPc), bis (diisobutyl-octadecylsiloxy) silicon 2, 3-naphthalocyanine (isoBOSINC), octabutoxy-phthalocyanine germanium IV (GePc), rhodamine 101 (Rh-101), rhodamine 110 (Rh-110), rhodamine 123 (Rh-123), rhodamine 19 (Rh-19) ), rhodamine 560 (Rh-560), rhodamine 575 (Rh-575), rhodamine 590 (Rh-590), rhodamine 610 (Rh-610), rhodamine 640 (Rh-640), rhodamine 6G (Rh-6G), rhodamine 700 (Rh-700), rhodamine 800 (Rh-800), rhodamine B (Rh-B), sulforhodamine 101, sulforhodamine 640, sulforide ina B, coumarin 1, coumarin 2, coumarin 4, coumarin 6, coumarin 6H, coumarin 7, coumarin 30, coumarin 47, coumarin 102, coumarin 106, coumarin 120, coumarin 151, coumarin 152, coumarin 152A, coumarin 153, coumarin 311, coumarin 307, coumarin 314, coumarin 334, coumarin 337, coumarin 343, coumarin 440, coumarin 450, coumarin 456, coumarin 460, coumarin 461, coumarin 466, coumarin 478, coumarin 480, coumarin 481 , coumarin 485, coumarin 490, coumarin 500, coumarin 503, coumarin 504, coumarin 510, coumarin 515, coumarin 519, coumarin 521, coumarin 522, coumarin 523, coumarin 535, coumarin 540, coumarin 540A, coumarin 548, 5-ethylamino- 9-diethylaminbenzo [a] -phenoxazinium (EtNBA), 5-ethylamino-9-diethylaminbenzo [a] phenothiazinium (EtNBS), 5-ethylamino-9-diethylaminobenzo [a] phenenelenazinium (EtNBSe), chlorpromazine, chlorpromazine derivatives, derivatives of chlorophyll, bacteriochlorophyll derivatives, metal ligand complexes, trichloride s (2,2'-bipyridine) ruthenium (II) (RuBPY), tris (2,2'-bipyridine) rhodium (II) dichloride (RhBPY), tris (2,2'-bipyridine) dichloride platinum (II) ) (PthPY), pheophorbide a, merocyanine 540, Vitamin D, 5-amino-levulinic acid, photosan, chlorin e6, ethylene diamine chlorin e6, mono-L-aspartyl chlorin e6, and N-blue derivatives of phenoxyzine, stilbene, stilbene derivatives and 4- (N- (2-hydroxyethyl) -N-methyl) -aminophenyl) -4 '- (6-hydroxy-xylsulfonyl) -stilbene (APSS). It is also more preferred that the at least one photoactive molecular agent is at least one photoactive biogenic molecular agent that is specific for a particular material or tissue within the particular volume of material, plant or animal tissue, even more preferred than the less a bioactive photogenic biogenic agent includes a segment selected from the group consisting of DNA, RNA, amino acids, proteins, antibodies, ligands, haptens, carbohydrate receptors or complexing agents, lipid receptors or co-binding agents, protein receptors or complexing agents, chelants, and encapsulating vehicles and it is still more preferred that the at least one bioactive photoactive biogenic agent further includes a segment that is photoactive when exposed to sufficient light to promote simultaneous excitation of two photons. In yet another embodiment of the present invention, the step of treating the particular volume of the material, plant tissue or animal tissue with sufficient light to promote simultaneous excitation of two photons of the at least one photoactive molecular agent contained in the particular volume of the material , vegetable tissue or animal tissue, also includes the stage of modulating light coming from a light source with a particular type of modulation, with which a modulated light is produced and the step of treating the particular volume of the material, plant or animal tissue with sufficient modulated light to promote a simultaneous excitation of two photons of the at least one photoactive molecular agent contained in the particular volume of the material, plant tissue or animal tissue. It is also preferred that the present invention further includes the steps of demodulating the energy signal detected with the particular type of modulation and producing a demodulated energy signal which is characteristic of the particular volume of the material, plant or animal tissue. It is more preferred that the step of demodulating the energy signal detected with the particular type of modulation includes demodulating the energy signal detected at a frequency twice that of the particular type of modulation, whereby the second harmonic of the particular type of modulation is detected. modulation. It is also more preferred that the demodulated energy signal that is characteristic of the particular volume of material, plant tissue or animal tissue represents a change in longevity of the at least one photoactivated molecular agent present in the particular volume of the material, plant tissue or animal tissue.

BRIEF DESCRIPTION OF THE DRAWINGS The above features, advantages and other features of the invention will be further appreciated from the following detailed description of preferred embodiments of the invention, in conjunction with the drawings. Figure 1 shows exemplary diagrams of energy levels for linear and non-linear optical excitation. Figure 2 shows the relationships between incident power distribution and excitation efficiency for excitation of a single photon and excitation of two photons. Figure 3 shows an example of absorption spectrum for animal tissue that covers the ultraviolet spectral region up to near the infrared spectral region. Figure 4 shows a diffusion spectrum for animal tissue that covers the ultraviolet spectral region up to near the infrared spectral region. Figure 5 shows the general trends in the optical diffusion and absorption properties of tissue for light of short wavelengths and incident long wavelengths.

Figure 6 compares optically induced excitation regions in tissue when single-photon and two-photon excitation methods are used. Figure 7 shows typical linear excitation properties of a diagnostic agent in solution. Figure 8 shows typical properties of non-linear excitation of a diagnostic agent in solution. Figure 9 shows a two-photon excited fluorescence photograph of the coumarin dye molecule 480 evenly distributed throughout the whole in an artificial tissue. Figure 10 shows a two-photon excited fluorescence photograph of the coumarin dye molecule 480 evenly distributed throughout a tumor sample. Figure 11 shows a diagram of a specific preferred embodiment of the present invention for imaging endogenous or exogenous imaging diagnostic agents. Figure 12 shows a diagram of an alternate preferred embodiment of the present invention for imaging endogenous or exogenous imaging diagnostic agents.

Figure 13 shows a diagram of a second alternate preferred embodiment of the present invention for forming videographic images of surface features.

Detailed description of the drawings The invention described herein utilizes the unique physical properties of nonlinear optical excitation of molecular agents to effect enhanced spatial control over the photoactivation of those molecular agents. In addition, nonlinear optical excitation has been shown to have additional advantages during photoactivation of medical diagnostic agents and other agents, including reduction of collateral excitation and damage along the excitation path, reduction in exposure to harmful optical wavelengths and reduction of interference from the absorption and diffusion processes that originate from the medium surrounding the excited agent. The fundamental importance of the invention described in this description is based on the use of simultaneous two-photon and non-linear optical excitation processes to photoactivate at long distance one or more molecular diagnostic agents with a high degree of spatial control and improved depth of penetration. These molecular agents can be exogenous agents added to the system under examination or they can be endogenous components of the system. Examples of exogenous diagnostic agents include various psoralen derivatives, while examples of endogenous agents include aromatic amino acids and nucleic acids. By focusing a beam of optical radiation on a sample under examination, the diagnostic agent can be excited in a location practically limited to the confocal region of the focused beam. The confocal region, Zs, is defined as the zone that extends a distance of 2pw02 / ?, where w0 is the diameter of the thinning or minimum waist of the beam and? is the wavelength of optical radiation. By contrast, when linear excitation methods are used, the excitation occurs substantially along the entire optical path, making the spatial location of excitation considerably less defined. In this way, the use of the two-photon excitation process greatly increases the excitation resolution along the optical path. Furthermore, since the excitation is effected at long wavelengths with respect to corresponding linear excitation processes, the diffusion and absorption of the excitation energy is greatly reduced. For optically dense and coarse samples, such as human tissue, this means that the excitation of two photons at considerably greater depths than is possible using linear excitation methods is possible. It is not necessary that the emitted light of the diagnostic agent be detected or image-formed directly without diffusion, since the spatial information relating to the origin of the emitted light is encrypted by and can be correlated to the focus of excitation. By moving the location of this focus relative to the sample, a bi or three-dimensional image of the emitted light can be developed. Also, by modulating the excitation light and using an appropriate demodulation method on the detection apparatus, the rejection of diffused excitation light and other interferences can be significantly improved. The present invention is primarily directed to the detection and formation of images of disease in vivo and other tissue characteristics, such as cancer in human breast. However, it will be apparent once the invention has been fully described that the methods and apparatus described herein have numerous additional applications and that these methods and apparatus can be applied to the field of two-photon laser scanning microscopy, as described by Denk et al., to achieve important improvements in the operating characteristics of such instruments. To begin this description, a review of the fundamental physics that supports linear and nonlinear optical excitation will be useful.

Comparison of linear and non-linear excitation - formulation of energy level diagram Figure 1 shows typical diagrams of molecular energy level for various linear and non-linear optical excitation processes. In this representation, which consists of simplified Jablonski diagrams, the vertical direction corresponds to a change in energy, while the horizontal direction represents the sequence of events and progresses from left to right. Solid horizontal lines represent quantum levels of mechanically permitted molecular energy, while horizontal dotted lines represent virtual levels of prohibited energy. Quantum levels of mechanically permitted molecular energy are of relatively long longevity and the probability of excitation of a molecule by energy absorption, such as that provided by absorption of a single photon of appropriate energy, is high. Virtual energy levels can be achieved through a variety of excitation processes, but in contrast to the allowed molecular transitions, they have extremely short longevity (in the order of 10"15 s, as predicted by the Heisenberg uncertainty principle). ), making them important only under special excitation conditions.The straight arrows in the Jablonski diagrams represent radiative energy transfer processes: the ascending arrows indicate energy absorption, while the descending arrows represent radiative emission, such as fluorescent or phosphorescent emission. The curved arrows represent non-radiative energy transfer processes, such as vibrational relaxation.The vertical length of the straight or curved arrow is proportional to the energy absorbed or emitted in a given process.For the first diagram Jablonski shown in the Figure 1, the excitation of a single photon up to a permitted energy level 2 occurs during the absorption of a photon 4 having sufficient energy to directly promote the molecule from a first allowed electronic energy level 6 (generally the lowest electronic energy level, or minimum energy state, indicated as ) to a second allowed electronic energy level 8 that has a higher overall energy level (represented here as the S2 state). Note that there may be several higher permitted electronic energetic levels at which excitation may occur and that these are commonly indicated by Si, S2, and so on as they increase their energy. The nomenclature Si indicates a singlet electronic energy level that conforms to the Pauli exclusion principle, where the turns of all the electrons are coupled and these coupled electron turns are opposite each other. Excited triplet states 10 may also be possible for some molecular systems, with the example denoted here as Ti. The triplet states differ from the singlet states in which the turns of all the electrons are coupled, except two. Each level of allowed electronic energy (singlet or triplet) can be further subdivided into a set of discrete vibrational levels 12; each of these discrete vibrational levels 12 can be further subdivided into a set of discrete rotational energy levels. Therefore, each permitted electronic energy level, So, Si, Ti, and so on, constitutes a complex band of allowed energy levels due to the large possible number of possible vibrational and rotating states. During the absorption of energy from a single photon 4 the molecule is promoted to a unique electronic and vibrational single level 14, sometimes indicated as a vibronic level. From this excited state the molecule can then undergo a rapid internal conversion 16, for example up to the allowed vibronic energy level allowed lower at the second allowed electronic energy level 8. This internal conversion 16 is usually very fast and occurs on a time scale in the order of 10"12 to 10-15 seconds., the excited molecule may undergo additional relaxation, such as through collisional 20 deactivation, to return to the initial and first energetic level 6. Alternate relaxation processes include fluorescent emission of a photon 21, which occurs directly from S to So and phosphorescence, which occurs after intersystem crossing 22 from a singlet state to a triplet state 10. Note that singlet-to-electronic singlet transitions, such as those shown for Si-S0, constitute mechanically permitted quantum transitions according to the Pauli exclusion principle . Conversely, transitions from a singlet state to a triplet state 10, such as Si - »Ti, are mechanically prohibited quantum transitions since the turns of the electrons do not remain coupled. However, the internal conversion probability is greater than zero for some molecular systems as a consequence of the relatively long longevity of the Si state compared to the constant intersystem crossing rate for these systems. The transition from the triplet state 10 back to the singlet state, such as Ti-So, can occur through the radiative process known as phosphorescent emission of a photon 24. Phosphorescence is generally characterized by a long radiative longevity compared to fluorescence due to the non-allowed nature of the process. An example of excitation of a single photon at a permitted energy level 2 is the promotion of the coumarin dye molecule from an electronic state of minimum energy to an excited electronic state through the absorption of a single photon 4 at 400 nm , followed by internal conversion 16 and subsequent fluorescent emission of a single photon 21 at 480 nm. In this example, the excitation probability is linearly related to the power of the incident optical radiation, in this way the excitation of a single photon up to an allowed energy level 2 is known as a linear excitation process. For the second Jablonski diagram shown in Figure 1, the excitation of a single photon to a virtual energy level 26 occurs during. the absorption of a photon 28 that has insufficient energy to promote the molecule directly up to an electronic energy level allowed 8 higher. Instead, the molecule is promoted to a virtual energetic level of very short life 30. This virtual energy level 30 will usually have a longevity in the order of 10-15 seconds. There will be virtually instantaneous re-emission 32 of the absorbed photon 28 from this virtual level 30 by means of processes such as elastic diffusion. An important example of this process is the diffusion of Rayleigh at 800 nm from coumarin during excitation with light at 800 nm. Another example is the diffusion of Raman, which occurs when the molecule returns to the different vibrational levels associated with the minimum energy or basal state. In these exemplary processes the excitation probability is also linearly related to the power of the incident optical radiation, so that the excitation of a single photon to a virtual energy level 26 is also known as a linear excitation process. For the final Jablonski diagram shown in Figure 1, the simultaneous excitation of two photons occurs up to an allowed energy or energy level 34 during the simultaneous absorption of a first of two photons 36 and a second of two photons 38. In this case the combined energy of the first of two photons 36 and the second of two photons 38 is sufficient to promote the molecule from a first allowed energy level 6 to a second allowed energy level 8. In general, none of the individual energies nor the first of two photons 36 nor of the second of two photons 38 is sufficient to directly promote this or any other permitted electronic transition. Instead of this, the first of two photons 36 promotes the molecule up to a low-energy virtual energy level 30. This is the same level of virtual energy as that shown in the second Jablonski diagram. Before remission 32 can occur from the virtual energy level 30, the second of two photons 38 immediately promotes the molecule to a second level of allowed electronic energy 8. The result is excitation that is equivalent to that achieved using linear excitation of a single photon up to an allowed energy level 2. Note that the first of two photons 36 and the second of two photons 38 can be of equal or different energy. Also, the instantaneous irradiation, or W m of the incident excitation light must be relatively high to provide a significant efficiency in the absorption of the second of two photons 38 before the virtual energy level 30 suffers relaxation 32 back to the first and original level of electronic energy allowed 6 In fact, because the longevity of the virtual energy level 30 is in the order of 10-15 seconds, normally pulsating excitation sources having very high peak powers are used to effectively stimulate these processes; such sources are often preferable because they are capable of providing large quantities of photons to the excited molecule during the brief longevity of the virtual energy level 30. Once the molecule has been promoted to the second allowed level of electronic energy 8, it can suffer a rapid internal conversion 16, followed by additional relaxation, such as through collisional deactivation 20, fluorescent emission of a photon 21, or intersystem crossing 22 to a triplet state 10. In the latter case, the transition of the triplet state 10 of return to the state of minimum singlet energy 6, can occur by means of phosphorescent emission of a photon 24. It is notable that the simultaneous excitation of two photons shares characteristics of both excitation of a single photon up to a permitted energy level 2 and excitation of a single photon up to a virtual energy level 26, specifically in which a virtual energy level 30 It plays an important role in the promotion of the molecule from the state of minimum energy to the excited state and that once promoted to an excited energy level the molecule can undergo photochemical and photophysical processes that are identical to those that result from excitation of a single photon. up to a permitted energy level 2. An example of the simultaneous two-photon excitation process is the promotion of the coumarin dye molecule from an electronic state of minimum energy to an excited electronic state by means of the simultaneous absorption of two photons at 800 nm, followed by emission of a fluorescent photon at 480 nm. Due to the well-known quadratic dependence on instantaneous photon irradiation, the simultaneous excitation of two photons up to a permitted energy level 50 is also known as a non-linear excitation process. The important differences between linear and non-linear excitation processes are identified in the next section. Note that in addition to the exemplary energy level diagrams shown in Figure 1, many other possible transitions and conditions of energy levels are possible, depending on many factors, including the characteristics of the molecular system, its surrounding environment and the particular energies of the forms of energy absorbed and released. Once a molecule has been promoted to an excited state, a variety of physical or chemical processes can occur, including luminescent emission of a photon, photochemical transformation, such as isomerization or oxidation or photoionization. Pretentiously, it is thought, that the fundamental properties of the excited state and its surrounding environment are what determine the final destination of the molecule.

Once excited, the mechanism responsible for promoting the molecule to the excited state has no major impact on this fate since the excitation process does not directly impact on the subsequent properties of the excited molecule or its environment. Therefore, a molecular diagnostic agent that works well under single-photon excitation conditions can be expected to exhibit similar behavior under two-photon excitation conditions.

Comparison of linear and non-linear excitation - power dependence and spatial effect When light interacts with a molecular system, it induces a polarization that is proportional to the linear susceptibility multiplied by the magnitude of the applied electric field. When this electric field is very intense, the system can not be described so easily and higher interaction terms must be included in the description of induced polarization. The simultaneous excitation of two photons is known as a nonlinear process because it occurs when the electromagnetic fields from two photons are combined by means of these higher order terms, specifically the imaginary portion of third order susceptibility, (3) ", to induce an electronic transition This is another way of describing the non-linearity of simultaneous absorption of two photons, that is, the molecular system reacts non-linearly with the intensity of the electromagnetic field.In contrast, single-photon excitation processes can to be described by means of linear susceptibility and are linear with excitation power Note that the cross section for simultaneous excitation of two photons is usually about one hundred thousand times smaller than that for a single photon equivalent excitation process This is due to the low probability that two photons interact simultaneously e with a molecule during the longevity of the extremely brief virtual energy level. However, the availability of optical excitation sources capable of providing extremely high peak powers, such as synchronized type lasers, can greatly improve the impact of this low efficiency, increasing the instantaneous incident powers and therefore greatly increasing the effective efficiency of the simultaneous excitation of two photons. For example, when continuous wave excitation is used the excitation efficiency of two photons for a particular molecular system may be smaller than that achieved with single-photon excitation. However, if the same average optical power is emitted in the form of a train of very short pulses, the change in product of the peak and average powers can change this ratio so that it is close to the unit. The non-linear nature of the simultaneous excitation of two photons can be exploited to achieve a significant difference in the spatial properties of simultaneous excitation of two photons compared to linear excitation. For example, Figure 2 shows that the excitation efficiency profile of a single photon 40 and the simultaneous excitation efficiency profile of two photons 42 differ significantly as a function of the beam intensity profile 44 when a light beam laser 46 focuses on a material 50. This material 50 could be a laser dye solution held between the walls of a spectrophotometer vessel 52. Another example of this material 50 could be human tissue of underlying skin. Focusing 48 of the laser light beam 46 with a lens 54 produces a beam intensity profile 44 that varies as a function of the distance through the sample 50, reaching a maximum level at the center of the focus 56 as predicted by the classical Gaussian optical theory. For a single-photon process, the linear relationship between beam intensity (or incident power) and excitation efficiency results in a single-photon excitation efficiency profile 40 which follows in a linear fashion the beam intensity profile 44. In contrast, for the simultaneous process of two photons, the non-linear relationship between beam intensity (or incident power) and excitation efficiency results in a simultaneous two-photon excitation efficiency profile 42 which follows the square of the beam intensity profile 44. Thus, the focusing 48 of the laser light beam 46 can be used to significantly limit the extent of the excitation to a small focus area when the simultaneous excitation of two photons is used. In contrast, when linear excitation is used, the excitation occurs substantially along the entire optical path, making the spatial location of the excitation considerably less defined.

Comparison of linear and non-linear excitation - absorption and diffusion effects While the cross section for simultaneous excitation of two photons may be considerably smaller than that observed with excitation of a single photon, the simultaneous excitation use of two photons may be favorable for the excitation of a single photon under many conditions due to the lower absorption of matrix and optical diffusion of optical radiation of longer wavelength. For example, Figure 3 shows an absorption spectrum 58 for animal tissue, such as human dermis or liver, which covers ultraviolet (UV) spectral region to the near infrared (NIR) spectral region. Figure 4 shows a diffusion spectrum 66 for animal tissue, such as human dermis or liver, under similar conditions. Specifically, Figure 3 demonstrates how higher energy photons 60, such as those used for linear excitation of diagnostic agents, can undergo considerably greater tissue absorption than lower energy photons 62, such as those used for non-linear excitation of low-energy agents. diagnosis. For example, human skin very strongly absorbs higher energy photons 60 to 400 nm, but is relatively transparent to lower energy photons 62 to 800 nm. This is a consequence of the relatively high natural absorbance of higher energy photons 60, having ultraviolet or visible wavelengths, by pigments, proteins, and genetic materials, among other natural components of the skin. Note also the relationship between excitation energies and the emission wavelength 64 of the diagnostic agent. Regardless of whether higher energy photons 60 or lower energy photons 62 are used to excite the agent, the emission wavelength 64 will occur at an energy that is determined by the agent, not by the excitation method applied to the agent. Figure 4 further demonstrates how higher energy photons 68 can undergo considerably greater tissue diffusion than lower energy photons 70. Any optically dense medium, such as human skin, will diffuse higher intensity photons 68 at wavelengths with greater intensity. visible or ultraviolet, for example at 400 nm, but will show much lower diffusion for lower energy photons 70 at NIR or infrared wavelengths for example at 800 nm. Note that as shown first in Figure 3, Figure 4 shows that the emission wavelength 72 of the diagnostic agent will typically fall between that of the higher energy photons 60 and that of the lower energy photons 62. These differences important in optical properties have many important consequences. First, the absorption of higher energy photons and short wavelengths 60 per tissue can result in undesirable tissue damage. In contrast, negligible effects can be experienced under irradiation with lower energy photons 62, such as NIR light, even though the optical power of NIR light is many times greater than that of UV or visible radiation. Second, the inherently high absorption and diffusion of higher energy photons 68 per tissue can produce very shallow tissue penetration depths, while lower energy photons 70 generally have larger penetration depths. In view of the fact that the higher energy photons 60 diffused will induce the emission of diagnostic agents along their diffusion trajectory, higher energy photons 60 that handle penetrating tissue will tend to produce a diffusion emission zone that extends perpendicularly. to the excitation path; but due to the quadratic dependence on the excitation of two photons, irradiation with lower energy photons 62 will produce a more precisely defined excitation pattern which is not defocused by the presence of diffused lower energy photons 62. Therefore, illumination and subsequent detection of features below the surface is difficult or impossible when using higher energy photons 68, such as those in the UV or visible spectral regions; In contrast, illumination and subsequent detection of features below the surface is much easier when using lower energy photons 70, such as those in the NIR or IR spectral regions. Note also that the emitted light of the diagnostic agent can be absorbed and diffused in an important way by the tissue or other optically dense medium under examination. However, for satisfactory detection of the emitted light, it is only necessary for a small fraction to make its way to a detector. The large extent to which this emitted light can be diffused implies that sophisticated methods are required to differentiate emitted light produced by an agent excited from diffused light and other optical or instrumental noise sources. This last consideration is the subject of a later section. These important differences in depth absorption and penetration properties for higher energy light and lower energy light are schematically shown in Figure 5. When UV light or visible light 74, for example light at 400 nm, strikes human tissue 76, most of the optical energy is absorbed 78 and diffuses 80 immediately into the outermost layers 82, such as the epidermis and the dermis. Absorption 78 can occur due to the excitation of certain molecules in the cells of this tissue 76, such as those that make up the genetic material in the cell nucleus and can initiate a variety of collateral photochemical changes in these cells at the site of this absorption 78. These collateral photochemical changes may include irreversible genetic damage and induction of cancer. Therefore, the optical penetration depth is low and the potential for induction of collateral damage is high for excitation with UV or visible light 74, such as that conventionally used for linear excitation of diagnostic agents. In contrast, light NIR or IR 84, for example at 800 nm, will experience much less absorption and diffusion 80 by tissue 76. The overall depth of penetration will be much greater and the extent of collateral damage to cells will be substantially less. Therefore, if the long wavelength excitation light is used in a two-photon excitation process to replace excitation of a single photon of higher energy, it will be possible to photoactivate specific diagnostic agents present in deep tissues using relatively long wavelengths not harmful that have great depths of penetration. In addition, the outstanding properties of nonlinear excitation shown in Figure 2 have additional implications when coupled with the inherent non-damaging nature and high penetration depths possible with the use of NIR light. For example, Figure 6 compares the characteristics of depth of penetration and spatial location expected for excitation of a single photon 86 and simultaneous excitation of two photons 88 of imaging agents present in a subcutaneous tumor 90. The excitation of a single photon 86 produces an excitation zone 92 that extends substantially along the entire optical path and has no significant specificity. Note that the efficiency of the excitation of a single photon 86 will vary along the optical path due to absorption and diffusion, with more can 94 near the point of introduction of optical radiation and rapidly decreasing 96 along the trajectory optics. Note also that the potential for induction of collateral photodamage will follow this same trend. Therefore, the excitation of a single photon produces an extended excitation zone 92 that can not be effectively delimited to a finite volume, specifically in deep tissues. Also, significant collateral damage can occur throughout the surrounding tissues 98 and especially in the superficial tissues 100. If the excitation of a single photon 86 is focused, the excitation zone 92 will be slightly improved at the focus 102. Note, however, that this excitation zone 92 may not extend throughout the trajectory in the tumor 90 if the UV or visible light used for single-photon excitation 86 it is absorbed or diffused in an important way before reaching tumor 90. In contrast, the use of simultaneous excitation of two NIR photons 88 produces a precisely defined remote excitation zone 104 which is located practically in focus 106 as a consequence of the non-linear intrinsic properties of this excitation method. Furthermore, due to the reduced absorption of NIR light, the collateral damage to the surrounding tissues 98 and especially to the surface tissues 100 is decreased. And as a consequence of the combined low absorption and diffusion of NIR light, it is possible to effectively test locations much deeper than those possible using UV wavelengths or visible wavelengths.

Examples of linear and non-linear excitation of imaging diagnostic agents The linear excitation of a diagnostic agent in solution is shown in Figure 7. In this example, laser radiation at 442 nm was used to excite a diluted solution of the FITC dye molecule in methanol. The laser beam emitted from a continuous wave helium: cadmium laser was focused through a 20x microscope objective in a spectrophotometer container containing the dye solution and stimulated an elongated and diffuse emission pattern in The dye. This example clearly shows that the emission occurs along the entire optical path and that a diffuse halo attributable to stimulation of the dye by diffused laser light surrounds the primary excitation path. In contrast, Figure 8 demonstrates remote and highly localized photoactivation of a diagnostic agent using simultaneous excitation of two photons. "In this example, laser radiation at 730 nm was used to excite a diluted solution of the coumarin dye molecule 480 in methanol. Specifically, the NIR output of a titanium laser: sapphire type synchronized, which emitted a continuous wavelength train of 730 nm, < 200 fs pulses of light at a pulse repetition frequency of 78 MHz in a beam of approximately 1 mm in diameter was focused through the same 20 x objective microscope lens in a spectrophotometer container containing the dye solution. Figure 8 clearly shows that the fluorescence response from the dye molecule is limited to the focus of the NIR beam. Due to the quadratic relationship between two-photon excitation and the instantaneous power of the laser, the stimulation at positions along the excitation path before and after the focus is negligible. No halo was observed either, although a small aberrant particle is observed in the photograph around the emission zone, attributable to the overexposure of the photographic film. Figure 9 demonstrates a highly localized remote photoactivation of a diagnostic agent present through an optically dense medium. This shows a two-photon excited fluorescence photograph of the coumarin dye molecule 480 completely distributed through an artificial tissue consisting of a block of agarose gelatin. Expanded NIR output of titanium laser: synchronized type sapphire that emitted a continuous wavelength train of 730 nm, < 200 fs pulses of light at a repetition frequency of 78 MHz pulses in a beam of approximately 1 mm in diameter, to produce a collimated beam of approximately 50 m in diameter using a beam expander telescope. This extended beam was then focused on the gelatin block using a biconvex singlet glass focal lens 50 mm in diameter and 100 mm focal length. The gelatin block was then placed in such a way that the focus of this 100 mm lens of f.l. fell in a position 40 mm inside the block. Figure 9 clearly shows that the fluorescence response of coumarin 480 is stimulated only at the focus of the NIR beam. Due to the quadratic relationship between the excitation of two photons and the instantaneous power of the laser, the stimulation at positions along the excitation path before and after the focus is negligible. Therefore, little or no collateral photoactivation of damage occurs outside the focus region. Also, because the NIR excitation light is only weakly diffused by the gelatin, the focus accuracy is maintained at deep penetration depths in the block. Note that the focus accuracy is determined by Gaussian optical properties; hence, the length of the confocal region is easily adjusted by changing the optical parameters used for beam expansion and subsequent refocusing. Similar results are obtained if an excitation process equivalent to a labeled tumor specimen is applied, as shown in Figure 10. This shows a two-photon excited fluorescence photograph of the coumarin dye molecule 480 evenly distributed throughout a block of mouse carcinoma tissue. As in Figure 9, a tightly localized site of activation is demonstrated, even for this sample having an extremely high optical density.

Excitation sources for two-photon excitation of diagnostic imaging agents The relatively low effective section for simultaneous two-photon excitation, which is usually about one hundred thousand times smaller than that for an equivalent single-photon excitation process, means that the optical excitation sources must be used to excite effective diagnostic agents. Optical sources that provide high peak power can be used to significantly improve the impact of this low efficiency, increasing the instantaneous incident power while maintaining modest average power levels. In fact, quasi-continuous wave synchronized type lasers are ideal, such as the titanium: sapphire type synchronized laser, to excite molecular diagnostic agents in optically dense samples, such as biological tissues. Specifically, such lasers are capable of delivering peak NIR powers greater than 10 kW, but in the form of very high repetition rates (pulse repetition rate> 25 MHz), ultra short (~ 200 fs of pulse duration), low energy pulses (~ 1 nJ per pulse); Average laser power partition (in the order of 10 mW to 2W) in a high-frequency train of ultra-short pulses provides an excitation beam that is extremely efficient for stimulating excited two-photon fluorescence but is essentially harmless for biological materials. The quasi-continuous output of synchronized type lasers or other high-speed repeating lasers are also quite compatible with various modulation methods, especially when the modulation is carried out at a frequency considerably below the pulse repetition frequency of the laser, since the nature Pulsing of the source can be ignored in the subsequent demodulation process. The specific example of the titanium: sapphire type synchronized laser is continuously adjusted over a wavelength band extending from about 690 nm to about 1080 nm, which corresponds well to a region of minimum diffusion and absorption for biological samples. The absorption of two photons in this band also corresponds to an important region of absorption of a single photon, from 345 nm to 540 nm, for many possible diagnostic agents; while the two-photon selection rules are often quite different from the corresponding single-photon selection rules, a strong absorption for the single-photon process may be indicative of significant two-photon absorption at wavelengths approximately twice that of the wavelength of a single photon. It will be clear that, in addition to the titanium: sapphire type synchronized laser, also, several other optical sources for excitation of diagnostic imaging agents are applicable. Especially important are diode lasers, Nd: YAG and Nd: YLF lasers, and parametric optical oscillators, amplifiers and generators. Pulsed diode lasers offer attractive performance as a result of their extremely high operating efficiencies and are available in a variety of wavelengths in the NIR. The synchronized type lasers, Nd: YAG and Nd: YLF provide reliable and efficient means to generate NIR excitation light at 1064 nm and at 1047 or 1053 nm, respectively. The synchronized type parametric oscillators, amplifiers and generators are capable of producing optical radiation that covers a band from about 500 nm to over 3000 nm; the availability of wavelengths from 1000 nm to 1800 nm provides a practical means for excitation diagnostic agents that use light in an extraordinarily low diffusion and absorption tissue band (ie, those that have single photon absorption bands) in band lengths greater than 500 nm).

Also, several other synchronized type lasers have application, including argon ion lasers, krypton ion lasers, helium-neon lasers, ruby lasers, Nd: YAP, Nd: YV04, Nd: Glass, and Nd lasers: CrGsGG; regeneratively amplified lasers; Lasers Cr: LiSF; Er: YAG lasers; F-center lasers; Ho lasers: YAF and Ho: YLFb and copper vapor lasers. Several continuous wave lasers can also be used but with considerably lower efficiency than that achieved using pulsed lasers.

Detection of excited emission of two photons from imaging diagnostic agents The spatial information regarding the origin of the light emitted from a two-photon imaging diagnostic agent is encrypted by means of and can be correlated to the excitation focus. This is in complete contrast to single-photon excited excitation methods, including those based on photon migration, where the diagnostic imaging signal should be carefully unwound from the emission light generated at throughout the entire excitation and the emission produced by diffused excitation light. Therefore, it is not necessary for the light emitted from the two-photon excited diagnostic agent to be detected or image-formed directly without diffusion. In fact, it is only necessary that a fraction of this emitted light is collected and detected in such a way that the collection and detection process does not distort the correlation between the detected signal and the point of origin of the emission. To understand the importance of the relationship between signal detection and the point of origin of the excited emission of two photons, it is useful to consider what happens to the light emitted immediately after the moment of emission. When images are formed in an optically dense sample, such as a biological tissue, the light from the excited two-photon imaging diagnostic agent will be emitted in an essentially isotropic manner. Some fraction of this emitted light will travel directly to a detector apparatus mounted far away from the emission point, while another fraction will travel a sinuous route to the detector apparatus as a consequence of one or more diffusion events that occur between emission and detection. If an attempt is made to form an image at a depth of 10 cm in a biological sample, the travel time for an emitted, non-diffused or ballistic photon (ie, the total travel time from the time of emission to the exit of the sample surface) will be approximately 0.3 ns; for a highly diffused emitted photon, this travel time could be as high as 3 to 10 ns. Thus, for maximum efficiency in this example, it may be desirable to integrate the totality of the emitted light for a sufficient period of time to capture most or all of the highly diffused ballistic photons. This implies that to form images at depths of 10 cm or less, an integration period of approximately 10 ns would be appropriate. If an image is to be generated by moving or exploring the location of the excitation focus relative to the sample, the above analysis implies that the excitation point should not move more frequently than every 10 ns. In fact, practical limitations in the exploration processes and mechanisms, combined with signal-to-noise arguments with respect to minimum resting or waiting times and the possible additional use of modulation methods, force the exploration to be carried out using higher waiting times. at 1 μs. In this way, for intensity-based image formation with waiting times greater than 1 μs and possible modulation frequencies of 1 MHz or less, it makes little difference where the detector is located as long as it is located in such a way that it can collecting a significant portion of ballistic and diffused light (the choice of the location of the detector in relation to the point of origin of emission and therefore the length of time introduced due to the optical delay, has little or no effect on the ability to correlate the detected signal with its origin due to the short travel time relative to other measurement parameters). Therefore, it will be clear that the detector can be located in such a way that it comprises an epi-illumination configuration with the excitation beam, or that it can be located external to the excitation beam. It is important to note that the epi-illumination configuration (or other possible excitation and collinear detection configurations) decrease potential paralax losses for detection of surface objects or near the surface, but such configurations are more susceptible to interference from elastically diffused light or reflected excitation light. Paralax losses can be minimized for external detection configurations by actively orienting the detection system in such a way that it keeps record consistent with the excitation point, using multiple detection assemblies that are individually optimized for light collection emitted from different areas within the sample in such a way that paralax losses are minimal. The discussion regarding the detection of emitted light from two-photon excited image-forming diagnostic agents has focused to this point on intensity-based methods, where an image can be constructed by correlation of detected emission intensity with the excitation location for multiple excitation points through a sample. However, intensity-based methods are not always optimal, because they are susceptible to a variety of complications, including: • Variations in the diffusion and absorption of excitation light due to heterogeneities in the sample - heterogeneities, such as areas of non-normal optical density, which are located between the source of excitation and the excitation target point can be translated into unanticipated differences at the level of effective excitation at the excitation target point. The aberrations caused by this phenomenon can be improved by acquiring data along several excitation trajectories that are affected at different extents by this heterogeneity, followed by subsequent unwinding of the resulting multiple data sets, but this can be difficult or impossible for some samples. • Variations in the diffusion and absorption of emitted light due to heterogeneities in the sample - heterogeneities, such as areas of non-normal optical density, which are located between the emission point and the detection system can be translated into unanticipated differences in the collection efficiency for light emitted from the excitation point. The aberrations caused by this phenomenon can be improved by acquiring data along several collection trajectories that are affected in different extents by this heterogeneity, followed by subsequent unwinding of the resulting multiple data sets, but this can be difficult or impossible for some samples. • Variations in the concentration or local environment of diagnostic imaging agents that are not correlated directly with form or function - it is assumed in the formation of intensity-based images that changes in emission level can be correlated through a sample with the physiological or structural organization of the sample. However, if the imaging agent is not properly distributed throughout the sample, or if other factors such as heterogeneity in the local environment within the sample, affect the emission of the imaging agent so that it does not can be correlated with form or function, then it becomes more difficult to obtain useful data from the sample. The aberrations caused by this phenomenon can be improved by the use or design of imaging agents that are not susceptible to such factors, but this may be difficult or impossible for some samples. A detection approach that is less susceptible to optical heterogeneity of the sample could be based on the measurement of the change in longevity of the excited state rather than relying on the emission intensity. The excited state longevity is an intrinsic property of the excited state of a molecular agent and its immediate surrounding medium., and fortuitously the exact measurement of longevity are immune to all but the strongest variations in level of excitation and collection efficiency. A convenient means for measuring excited state longevity uses phase photometric methods to correlate phase changes between a modulated excitation source and the resulting emission signal for longevity. Specifically, the preceding discussion about photon travel times implies that phase photometric methods are applicable for forming images in optically dense media, especially for agents with longevity greater than 1 to 10 ns. Thus, if imaging diagnostic agents are used they have emission longevity that correlates with shape or function within the sample, such as fluorescence quenching of an image forming agent in the presence of oxygen or concentration of an agent image forming within a structure, then image formation based on the change in longevity instead of emission intensity becomes practical. Such longevity-based methods could have equal applicability to laser scanning microscopy and remote image formation of extended objects, such as a tumor in a human subject. Suitable collection devices for transduction of emission data based on phase or intensity include, but are not limited to, photomultiplier tubes, microchannel plate devices, photodiodes, avalanche photodiodes, load-bearing devices and arrays of load-bearing devices, devices of cargo injection and arrangements of charge injection devices and photographic films.

Noise reduction methods for recovery of excited emission of two photons from imaging diagnostic agents - modulation and second harmonic detection The inherently low efficiency of two-photon excitation processes can be translated into a very high proportion of diffused excitation light and without absorbing excited two-photon fluorescence emission. In addition, the importance of other possible interferences attributable to this very high level of excitation, including excited fluorescence of a single photon of the agent or other species present in the sample under study, Raman diffusion, and other phenomena, together with the need to eliminate interference from ambient light and other sources of electronic or optical noise, all indicate that a modulated excitation method coupled with appropriate demodulation of the detector signal should provide optimal discrimination against interference and improve the recovery of the analytical signal. In fact, the interference from the background reported by Denk et al. (U.S. Patent No. 5,034,613) could be eliminated in an important manner if suitable modulation and demodulation methods were used, including demodulation in the pulse repetition frequency of the laser; the use of such methods could significantly improve the performance (SNR) signal to noise of your microscope. In general, modulation can improve detection performance for virtually any measurement in one or more ways: (1) Rejection of continuous sources of background or noise - in the example of Denk's two-photon laser scanning microscope, the modulation of the excitation source with subsequent demodulation of the detector signal, using a device such as a synchronous amplifier (LIA) or a heterodino demodulator, could limit the response of the detection system to a band of frequencies closely related to the demodulation frequency. By controlling the phase sensitivity of this demodulation, additional discrimination could be achieved against signals that are not linked or closely coincident with the demodulation pattern. Therefore, by appropriate selection of modulation frequency and demodulation phase, interference from noise sources such as ambient light or electronic noise at specific frequencies, for example coming from a nearby electric motor, can be strongly rejected. This approach is equally valid for the formation of remote images of extended objects, such as tumor in a human subject. (2) Rejection of broadband sources or xx pink noise "- the measurement of environment, together with electronic devices and other devices used for any measurement, contribute to broadband noise, sometimes called pink noise, in any measurement The impact of this intrinsic noise can be greatly reduced through the use of limited detection methods.Specifically, for a given optical measurement, the observed signal voltage, Vseñai, is related to a sensing input current, in / produced by photons interacting with a detector, multiplied by the input impedance, tada, and the gain of the detection system, G, in accordance with the following: VERSION - 3-INPUT • ^ OUTPUT • G, (1) while the observed noise voltage, VRUIDO / can be approximated by the product of the noise current, the input impedance, the square root of the bandwidth optical or electronic, B, of the detection and gain system, in accordance with the following: NOISE - J-NOISE • Z] ENTRY B1 2. (2) Therefore SNR can be estimated from the ratio of these two voltages, (VSEÑAL / VRUIDO) • When using a conventional optical detector, such as a photomultiplier tube (PMT), to detect an unmodulated fluorescent signalR. , this detector will produce a certain level of signal along with a noise current. For an exemplary PMT, such as the Hamamatsu R928 (radiant anode sensitivity 7.4xl05 A / W), an optical input at a level of 10 pW produces an ISSUE of 7.4 μA. If this signal current is converted to voltage in a low noise amplifier having a gain of 100, an input impedance of 50 O, an input noise level of 5 nV /? / Hz, and a bandwidth of 1 MHz, the following signals will be produced: V £ SIGNAL = 7.4 μA. 50 O. 100 = 37mV; Vruid = 5 Nv / HZ • (107 Hz) 1/2 • 100 = 1.6 mV.

Note that Ohm's Law, or V = i • R, has been substituted for noise and impedance current shown in equation 2. Thus, for this exemplary bandwidth, SNR = 23. If this excitation energy is modulates, for example in sinusoidal form at 1 MHz with a 100% depth of modulation, the value of VSEÑAL will decrease to approximately 18.5 mV (assuming that this modulation is introduced by cyclic attenuation or another method of modulation based on loss that provides a loss overall 50% average power without changing the peak excitation power). But if the detection system uses limited demodulation of bandwidth at 1 MHz that has a bandwidth of 1 kHz, the pink noise will decrease much faster than the signal: Vruid = 5 Nv / HZ • (103 Hz) 1/2 • 100 = 16 μV, and the global SNR increases to approximately 1200. Thus, even when some signal strength is lost using many forms of modulation, the overall increase in SNR more than compensates for this loss. In addition, if there is any linear interference in the detector response, for example from ambient light leakage in the detector, the bandwidth detection scheme will detect this as an additional source of noise, at the same time the limited width scheme of band will reject this interference. Assume that the environmental leak produces a background signal of 1 μA in the PMT, which translates to 5 mV of background signal. For the unmodulated case, the optical trigger noise from this background, B, is equal to the square root of the total photons detected and SNR ~ S / (S + B) 1/2; this provides an estimated SNR of approximately 5.7. Notably, the SNR for the modulated case remains essentially unchanged. This analysis is equally applicable to laser scanning microscopy and the formation of remote images of extended objects, such as tumors in a human subject. (3) Rejection of linear interference in the modulation frequency - as a consequence of the inherently low efficiency of two-photon excitation, the proportion of excitation light that is not absorbed and diffused to two-photon excited fluorescence emission is usually very high. This includes linear interferences in the demodulation frequency that result from non-elastic and elastic diffusion as well as excited fluorescence from a single photon. Optical filtering is often used in an effort to distinguish spectrally two-photon emission from these optical background phenomena. Unfortunately, these interferences can be very difficult or impossible to eliminate using only spectral means. As an alternative to ignore these sources of residual interference, a common approach for the recovery of pure two-photon signal uses regression of the detected signal at several levels of excitation power against the excitation power level, so that the quadratic component Two-photon excited fluorescence can be mathematically extracted from linear interference; this makes use of a total fluorescence response model, Jf, given by: Jf = aJL + ßJ2 (3) where IL is the instantaneous excitation current, a is a proportionality constant for several linear effects, and ß is a proportionality constant for two-photon excited fluorescence. While this regression-based method is appropriate for use at the laboratory level, where the required number of measurements per unit of time is small, it is time-consuming, complicated and not practical as long as the total data acquisition must be minimized, such as in the case of optical image exploration scanned from multiple points. Much faster results can be obtained through the use of temporary rejection methods, such as second harmonic detection, which eliminates the need for multiple measurements at many power levels. Freeman et al. (RG Freeman, DL Gilliand and FE Lytle, * Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence, "Analytical Chemistry, 62 (1990) 2216-2219) describe second harmonic detection methods useful for the analysis of chemical samples, where the sinusoidal modulation of the excitation source is used to generate a signal at two times the modulation frequency that relates only to two-photon excited fluorescence.A synchronous amplifier referenced to the modulation frequency is used to recover the pure signal of two photons in the second harmonic of the modulation frequency.While the second harmonic fluorescence signal is only about 12% of the total two photon fluorescence produced, the improved rejection of linear interference more than compensates for the loss in the absolute signal level, resulting in an increase in the global SNR, therefore, the method of Second harmonic detection is ideally applicable to laser scanning microscopy and remote image formation of extended objects, such as a tumor in a human subject, as a consequence of its intrinsic efficiency in diffusion rejection and its high width potential of data band. These advantages mean that an imaging system that uses second harmonic detection can reliably obtain two-photon excited emission signals with minimum wait times at each point, and with the use of maximum excitation power for each measurement at every point. The above advantages for the use of modulation methods in the formation of two-photon excited diagnostic images are applied in the same way regardless of whether the data were acquired based on emission intensity measurement or excited state longevity. In fact, longevity measurements are the easiest and most sensitive measurements using photometric phase methods that are based on the determination of phase changes between a modulation waveform and the detected signal. Therefore, it is clear that modulation methods, including those based on second harmonic detection, have important utility in the efficient detection of excited fluorescence of two photons, where they serve to eliminate interference from environmental sources and instrumental noise as well as from diffusion phenomena and other phenomena that occur within the sample undergoing the examination. For optically dense media, such as human tissue, the extremely high proportion of unabsorbed and diffused excitation light to the excited two-photon fluorescent emission makes use of such vital methods. Therefore, for applications of clinical imaging or laser scanning microscopy, the use of modulation methods as described herein will always be advantageous.

Contrast agents in excited two-photon imaging - endogenous and exogenous agents The above discussion has shown that the non-linear two-photon excitation can be used to effect significant improvements in the specificity and depth of penetration for optically excitable molecular agents present in optically dense media and that the detection performance can be improved using coding and decoding methods in the respective processes of excitation and detection. The exceptional spatial location of possible excitation when using two-photon methods can be used to significantly improve the contrast at the excitation point. Once this localized excitation is effected, the analytical light emitted in this way can be detected using a variety of detection means. If this excitation point is forced to move in relation to the sample under study, for example by exploring the position of the focus relative to the sample or by exploring the position of the sample with respect to the focus, then a bi or three-dimensional image of the image can be generated. the sample making a correlation between the location of the excitation point and the emitted light produced by it. The useful contrast in this image, however, also depends on the existence of differences in the concentration or local environment of the molecular agents responsible for emission. These agents can be endogenous or exogenous to the sample and the imaging is ultimately based on contrasts in their localized emission properties that can be correlated to heterogeneity in structure or function within the sample. Therefore, it is also important to carefully consider the role of these contrast agents in the formation of non-linear diagnostic imaging. Various endogenous chromophoric agents may be useful for diagnostic imaging, particularly of diseased tissue. Due to structural or physiological differences between diseased and non-diseased tissues, between various internal substructures and organisms in higher animals, or between different ranges of healthy or near-healthy tissues, the concentration or local environment of natural chromophoric agents, such as aromatic amino acids, proteins, nucleic acids, stores (such as adenosine triphosphate) of cellular energy exchange, enzymes, hormones or other agents can vary in ways that are useful for probing structural or functional heterogeneity. In this way, these endogenous indicators of heterogeneity can be tested using non-invasive two-photon excitation. . Unfortunately, in many cases the specificity possible with such agents is inadequate to achieve significant diagnostic imaging, and thus exogenous agents must be added to the sample. Traditional exogenous agents are semi-selectively partitioned into specific tissues, organs or other structural units of a sample after administration. The route of administration of these agents is usually topical application or systemic administration. Under ideal conditions, these agents will be partitioned into or in some way concentrated on or within the structures of interest, or preferentially excluded from these structures. This concentration can be a consequence of topical application isolated directly on a surface structure, or through intrinsic differences in the physical or chemical properties of the structure that lead to the partition of the agent in the structure. The contrast between areas of high concentration and low concentration can therefore be used as bases to probe physiological or structural heterogeneity. Alternatively, exogenous agents may pass through the sample; If its emission properties, such as chromatic change, damping, or longevity, are sensitive to physiological heterogeneity, then these parameters of the contrast agent can be used as the basis for contrast in imaging. Because the emission properties of a molecular agent are determined by the fundamental properties of the excited state and its environment, the mechanism responsible for promoting the agent to the excited state has no significant effect on the emission properties of the excited state. Thus, a molecular or diagnostic agent that works well under single-photon excitation conditions can be expected to exhibit similar behavior under two-photon excitation conditions. In general, any contrast agent that is useful in excitation of a single photon can be used with two-photon excitation, where the improved control over the excitation site will serve to improve the resolution of the image. Appropriate contrast agents include many molecular agents used as biological inks or stains, as well as those used for photodynamic therapy (PDT). Normal TFD agents have tissue specificities that are generally based on the combined chemical and physical properties of the agent and the tissue, such as a cancerous lesion. These agents are efficient absorbers of optical energy and in many cases are luminescent. For example, • psoralen and its derivatives (including 5-methoxypsoralen [or 5-MOP]; 8-methoxypsoralen [8-MOP]; 4,5 ', 8-trimethylpsoralen [TMP]; 4'-aminomethyl-4,5 ', 8-trimethylpsoralen [AMT]; 4'-hydroxymethyl-4,5 ', 8-trimethylpsoralen [HMT]; 5- chloromethyl-8-methoxypsoralen, Angelicin [isopsoralen]; 5-methylangelicine [5-MIP]; and 3- carbetoxypsoralen); • various derivatives of porphyrin and hematoporphyrin (including hematoporphyrin derivative [HPD], Photofrin II, benzoporphyrin derivatives [BPD], protoporphyrin IX [Pp IX], hematoporphyrin dye ether [DHE], polyhematoporphyrin esters [PHE]; 13, 17-N, N, N-dimethylethyl ethanolamine protoporphyrinine ester [PH1008], tetra (3-hydroxyphenyl) -porphyrin [3- THPP], tetraphenylporphyrin monosulfonate [TPPS1], tetraphenylporphyrin disulfonate [TPPS2a], dihematoporphyrin ether, meso-tetraphenyl-porphyrin, and mesotetra (4N-methylpyridyl) porphyrin [T4MPyP]) together with several tetraazaporphyrins (including octa- (4-tert-butylphenyl) -tetrapyrazineporphirazine [OPTP]; tetra- (4-tert-butyl) phthalocyanine [t-PcH2]; (4- tert-butyl) phthalocyanatomagnesium [t4-PcMg]); • various phthalocyanine derivatives (including sulfonated chloraluminium phthalocyanine [CASPc]; chloraluminium phthalocyanine tetrasulfate [AIPcTS]; mono-, di-, tri- and tetrasulfated aluminum phthalocyanines [including ISISc, A1S2PC, AlS3Pc and AlS4Pc]; phthalocyanine silicon [SiPc IV]; phthalocyanine zinc (II) [ZnPc]; bis (di- isobutyloctadecylsilyloxy) silicon 2,3-naphthalocyanine [isoBOSINC]); and Ge (IV) -octabutoxy-phthalocyanine; • various rhodamine derivatives (including rhodamine-101 [Rh-101]; rhodamine-110 [Rh-110]; rhodamine-123 [Rh-123]; rhodamine-19 [Rh-19]; rhodamine-560 [Rh-560] ]: rhodamine-575 [Rh-575]; rhodamine-590 [Rh590]; rhodamine-610 [Rh-610]; rhodamine-640 [Rh-640]; rhodamine-6G [Rh-6G]; rhodamine-700 [Rh] -700]; rhodamine-800 [Rh-800]; rhodamine-B [Rh-B]; sulforhodamine 640 or 101; and sulforhodamine B); • various coumarin derivatives (including coumarin 1, 2, 4, 6, 6H, 7. 30, 47, 102, 106, 120, 151, 152, 152A, 153, 311, 307, 314. 334, 337, 343. 440, 450, 456, 460, 461, 466, 478, 480, 481, 485, 490, 500, 503, 504, 510, 515, 519, 521, 522, 523, 535, 540, 540A, 548); • various benzophenoxazine derivatives (including 5-ethylamino-9-diethylaminobenzo [a] -phenoxazinium [EtNBA]; 5-ethylamino-9-diethylaminobenzo [a] phenothiazinium [EtNBS], and 5-ethylamine-9-diethylaminobenzo [a] phenenelenazinium) [EtNBSe]); • Chlorpromazine and its derivatives; • various chlorophyll derivatives and bacteriochlorophyll derivatives (including bacteriochlorin a [BCA]); • various complexes of metal ligands, such as tris (2,2 '-bipyridine) ruthenium (II) dichloride (RuBPY); • pheophorbide a [Pheo, a]; Merocyanine 540 [MC 540]; Vitamin D; 5-amino levulinic acid [ALA]; photos; chlorin e6, ethylenediamide chlorin e6, and mono-L-aspartyl chlorin e6; feoforbida-a [Ph-a]; Nilo blue derivatives of phenoxyzine (including several phenoxazine dyes); • various charge transfer agents and radiative transfer agents, such as stilbene, stilbene derivatives and 4- (N- (2-hydroxyethyl) -N-methyl]) -aminophenyl) -4 '(6-hydroxyhexylsulfonyl) stilbene ( APSS); and • many other photoactive agents will generally become accumulated either at or near an application point or semi-selectively within a specific tissue due to differences in the physical or chemical properties of the tissue that lead to the dividing of the TFD agent into the tissue; once accumulated, such agents will become susceptible to two-photon excitation and their luminescence or other emission properties can be used to acquire image set data. Other photoactive agents that absorb light and are capable of subsequent energy transfer to one or more other agents can also be used, either alone or in conjunction with one or more corresponding agents that are capable of accepting this transferred energy and transforming it into a radiative emission.

Biogenic contrast agents in excited two-photon imaging Under ideal conditions, normal contrast agents derive objective specificity based on chemical or physical affinity for specific tissues. In this way, the partition of contrast agents within or in some way concentrated on or within tissues of interest. Unfortunately, this objective specificity is usually not perfect. In fact, it is desirable to have an improved method for increasing the specificity in the targeting selection of the agent. A means to achieve such an improvement in specificity is based on the use of specific biological identifications of structure, function or disease. For example, by coupling antisense oligonucleotide agents to one or more photoactive portions, such as FITC, new biogenic contrast agents are created that are capable of selectively labeling only specific cells, such as cancer cells containing complementary genetic coding. Furthermore, the basic approach easily extends to many genetic diseases or other diseases by changing the oligomeric code used for the biogenic probe. The use of two-photon activation makes this powerful approach possible to be applied using the combined biospecificity of the biogenic probe and the high spatial location inherent to the simultaneous photon activation process of two photons. In this way, a very high contrast and very high resolution of genetic imaging is possible using agents that are specifically selected for a particular organ, tissue or injury. An optimal design for biogenic probe uses one or more photoactive portions that have emission properties that change in complexation between the biogenetic agent and the target site. Specifically, changes in emission wavelength or longevity during complexation can be used to increase the sensitivity of the general method, since such changes will help to increase the contrast between areas containing complex agents and those containing non-complexing agents. An example is a biogenic agent based on a photoactive portion that is buffered until complex occurrence occurs, during which the occurrence emission becomes un-muffled. Another example is an agent based on an intercalating photoactive portion, such as psoralen, which is in captivity up to an antisense genetic sequence.; In the complexity between the antisense sequence and its target sequence, the intercalation of the photoactive portion makes it possible that it also leads to a chromatic change in the emission properties of the photoactive portion. It will be clear from the above discussion that targeting methods based on other biospecific media, such as immunological media, rather than only on genetic media, are also covered within the scope of the invention. Specifically, the agent specificity based on antigen-antibody methods, wherein an antibody probe that couples to a photoactive group, provides a powerful new means for diagnosis of disease and infection. Additional means to achieve biospecificity in targeting agent selection include, but are not limited to, use of ligands, haptens, carbohydrates, lipids or protein receptors or complexing agents before, chelators, and encapsulating vehicles, such as liposomes, fullerenes , crown ethers and cyclodextrins.

FIRST EXEMPLARY MODALITY OF THE INVENTION Accordingly, it is a preferred specific embodiment of the present invention to employ the output of a NIR source to induce simultaneous photoactivation of two photons of endogenous or exogenous imaging diagnostic agents present in a sample using light at a length of wave about twice that needed for the conventional photoactivation of a single photon. This preferred embodiment is shown in Figure 11. The NIR source 108 produces a NIR radiation beam 110 consisting of a rapid series of high peak power pulses of NIR radiation. For example, commercially available synchronized titanium-sapphire type lasers are capable of producing synchronized type pulse outputs with durations < 200 fs and pulse energies of approximately 20 nJ at pulse repetition frequencies above 75 MHz; this source produces a quasi-continuous light beam that has a relatively low average power (up to several watts) but high peak powers (in the order of 100 kW), which is continuously adjustable over a NIR wavelength band from about 690 up to 1080 nm. The train of pulses emitted by the NIR source 108 constitutes a NIR radiation beam 110 which is easily focused using normal optical means, such as reflective or refractive optics. The focused NIR beam 114 can then be oriented on a sample 116 to be imaged. The simultaneous photoactivation of two photons of the imaging diagnostic agent will be substantially limited to the confocal region 118 of the focused beam 114 due to the high level of instantaneous irradiation that is only present in the focus. The excitation light that is diffused 120 by the sample 116 will not have a sufficient instantaneous irradiation level for significant excitation of any diagnostic imaging agent that may be present in the confocal region 118. The light emitted 122 by molecules of agents Diagnosis of imaging present in the confocal region 118 will leave the confocal region 118 in a substantially isotropic manner. A part of the emitted light 124 is captured by a detection means 126, such as a photomultiplier tube, which is mounted in a position inside or outside the sample 116. This detection means 126 is equipped with a length selection means. wave 128, such as an optical bandpass filter, which serves to preprocess the captured portion of the emitted light 124 such that the selection means 128 rejects a large fraction of the elastically diffused light as a fraction passes through greater light in the length or wavelengths corresponding to that which is mainly the emission characteristic coming from the diagnostic agent. The signal then delivered 130 of the detection means 126 is captured by means of a processor means 132, whose primary purpose is to record the emission response from the image forming diagnostic agent as a function of the location of the confocal 118 region. that the location of the confocal region 118 to be scanned through the volume of the sample 116, a complete image of the sample 116 can be obtained by examining the content of the processor means 132 as a function of the location of the confocal region 118. This image can be used to identify areas of interest 134, such as subcutaneous tumors or other diseased areas.

SECOND MODALITY EXEMPLARY OF THE INVENTION As an alternative to this preferred embodiment, a modulation means may be incorporated in the general mode shown in Figure 11; the modulation means can be used to improve the overall performance of the imaging system, such as to improve the rejection of environmental or instrumental noise sources, to allow the recovery of excited emission of two pure photons in the second harmonic, or to facilitate the detection of light emitted using photometric phase approximations. Specifically, FIG. 12 shows that a modulation means 136, such as an electro-optic or acousto-optic modulator, a vibrator, or other means, located in such a way that it interacts with the NIR radiation beam 110 emitted by the NIR source 108 can be used for encoding the NIR radiation beam 110 with a modulation pattern that is recorded at the output of a modulator controller 138 that provides a control signal 140 for the modulation means 136. The modulated beam of NIR radiation 142 produced in this manner is then orientates on sample 116 as described above for Figure 11. The emitted two-photon excited light 144 produced in this manner will leave confocal region 118 in an essentially isotropic manner 144. However, in contrast to similar emitted light 122 previously described for Figure 11, this emitted light 144 will show a modulation that is essentially synchronous with the modulation of the modulated beam of r NIR 142, which in turn is synchronous with the control signal 140 delivered by the modulator controller 138. A part of the emitted modulated light 146 is captured by means of a detection means 126, such as a photomultiplier tube, which it is mounted in a position inside or outside the sample 116. This image detection means 126 is equipped with wavelength selection means 128, such as an optical band pass filter, which is used to process the captured part of the light emitted modulated 146 in such a way that the selection means 128 rejects a majority part of the elastically diffused light while passing a greater part of light at the wavelength or wavelengths corresponding to that which is mainly the emission characteristic coming from of the diagnostic agent. The signal modulated in this way delivered 148 from the image forming means 126 is captured by a processor means 150. The processor means 150 serves two primary purposes, firstly to demodulate the modulated signal thus delivered 148 from the medium detection 126 using a demodulation reference output 152 delivered by the modulator controller 138 and secondly to record the demodulated emission response from the image forming diagnostic agent as a function of the location of the confocal 118 region. both, by causing the location of the confocal region 118 to be scanned through the entire volume of the sample 116, a complete image of the sample 116 can be obtained by examining the contents of the processor means 150 as a function of the location of the confocal region 118. This image can be used to identify areas of interest 134 such as tumo subcutaneous or other diseased areas.

THIRD EXEMPLARY MODALITY OF THE INVENTION As a second alternative for this alternative embodiment, an unfocused NIR radiation beam can be used to illuminate surface features of a sample to provide a direct means of detection imaging. This is shown in Figure 13. Specifically, the output of a NIR source, such as the titanium: sapphire synchronized mode laser can be used to induce simultaneous photoactivation of two photons of endogenous or exogenous imaging diagnostic agents present in or near of the surface of a sample using light at a wavelength to approximately twice that required for conventional photon activation of a single photon. The NIR source 108 produces a NIR radiation beam 110 consisting of a rapid series of high peak NIR radiation pulses. This beam is modulated using a modulator means 136 located so as to interact with the NIR radiation beam 110 emitted by the NIR source 108. This modulator means 136 codes the NIR radiation beam 110 with a modulation pattern that is recorded at the output of a modulator controller 138 that provides a control signal 140 for the modulation means 136. The modulated beam of NIR radiation 142 produced in this manner is then out of focus using normal optical means, such as reflective or refractive optics 154, to produce a beam of divergent excitation 156 which is oriented on a sample 116 to be formed in images. Simultaneous photoactivation of two photons of diagnostic imaging agent present on or near the surface of the sample 116 produces excited two-photon excited light 144 having a modulation that is essentially synchronous with the modulation of the NIR radiation beam 142 , which in turn is synchronous with the control signal 140 delivered by the modulator controller 138. A portion of the emitted modulated light 146 is captured by means of an image forming detection means 158, such as a device array. charge trailer, which is mounted in a position outside the sample 116. This image detection means 158 is equipped with a wavelength selection means 128, such as an optical band pass filter, which serves to process the captured part of the modulated emitted light 146 in such a way that the selection means 128 rejects a larger part of the elastically diffused light at the same time that a part of the light passes or light at the wavelength or wavelengths corresponding to that which is mainly the emission characteristic from the diagnostic agent. The signal modulated in this way delivered 160 from the imaging detection means 158 is captured by a processor means 162. The processor means 162 serves two primary purposes, firstly to demodulate the modulated signal thus delivered 160 from the medium of image formation detection 158 using a demodulation reference output 152 delivered by the modulator controller 138 and secondly to record the demodulated emission response from the image forming diagnostic agent as a function of the location of the emission . Therefore, this alternating modlaity makes it possible to perform the direct viodegraphic imagery of surface features 164, such as cancerous skin lesions, based on spatial differences in excited emission of two photons across the illuminated surface of the sample 116 It will be understood that each of the elements described above, or two or more as a whole, may also find useful application in other types of constructions or applications different to the types described above. While the invention has been described and illustrated as a modality in a general method for improved selectivity in photoactivation of molecular diagnostic agents imaging agents, is not intended to be limited to the details shown, since it will be understood that several omissions, modifications, substitutions and changes in the forms and details of the illustrated method can be made and in its operation can be done by those experienced in the medium without departing in any way from the scope of the invention. For example, in the third exemplary embodiment, modulation and demodulation details may be omitted to produce a simpler image forming apparatus, although this exemplary modification could lead to a general reduction in imaging performance. Without further analysis, the foregoing will fully demonstrate the essence of the present invention that others can, applying common knowledge, easily adapt it to various applications without omitting characteristics that, from the point of view of the prior art, justly constitute essential characteristics of the generic or specific aspects of this invention. What is claimed as new and desired to be protected by means of a patent document is set forth in the appended claims.

Claims (55)

1. A method for forming images of a particular volume of material, wherein the material contains at least one photoactive molecular agent, characterized in that the method comprises the steps of: (a) treating the particular volume of the material with sufficient light to promote excitation of two photons of at least one of the at least one photoactive molecular agent contained in the particular volume of the material and photoactivating the at least one photoactive molecular agent in the particular volume of the material, thereby producing at least one molecular agent photoactivated, wherein the at least one photoactivated molecular agent emits energy; (b) detecting the energy emitted by the at least one photoactivated molecular agent and (c) producing a detected energy signal that is characteristic of the particular volume of the material.

2. A method according to claim 1, further characterized in that the material is plant tissue or animal or human tissue.

3. A method according to claim 2, further characterized in that the animal or human tissue is not present in a living human or animal body.

4. A method according to any of claims 1 to 3, further characterized in that the excitation of two photons is simultaneous excitation of two photons.

5. A method according to any of the preceding claims, for spatially selective imaging of a particular volume of tissue, characterized in that the tissue contains a photoactive agent, the method comprising the steps of: orienting light to the particular volume of tissue of a way that promotes excitation of two photons of the photoactive agent in the particular volume of tissue, so that the photoactivated agent emits energy, detecting the energy emitted by the photoactivated agent; producing a detected energy signal that is characteristic of the particular volume of tissue and forming images of the particular volume of tissue based on the detected energy signal.

6. A method according to any of the preceding claims, further characterized in that the energy is radiant energy.

7. A method according to any of the preceding claims, further characterized in that the photoactive agent is an endogenous agent.

8. A method according to any of claims 1 to 6, further characterized in that the photoactive agent is an exogenous agent.

9. A method according to any of the preceding claims, further characterized in that the method further includes a first step of treating the material with at least one photoactive molecular agent, wherein the particular volume of the material retains at least a portion of the at least one photoactive molecular agent.

A method according to any one of the preceding claims, further characterized in that the at least one photoactive molecular agent comprises at least one bioactive photo-active molecular agent that is specific for a particular substance within the particular volume of material.

11. A method according to claim 10, further characterized in that the particular substance is a particular tissue.

12. A method according to claim 11, further characterized in that the at least one bioactive photoactive biogenic agent includes a segment which is photoactive when exposed to sufficient light to promote a two-photon excitation.

13. A method according to any of claims 10 to 12, further characterized in that the at least one bioactive photoactive biogenic agent includes a selected segment of DNA, RNA, amino acids, proteins, antibodies, ligands, haptens, hydrate receptors of carbon or complex agents before, lipid receptors or complex agents before, protein receptors or complex agents before, chelators and encapsulating vehicles.

A method according to any of the preceding claims, further characterized in that the at least one photoactive molecular agent is selected from the group consisting of psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) , 4, 5 ', 8-trimethylpsoralen (TMP), 4'-aminomethyl-4, 5", 8-trimethylpsoralen (AMT), 5-chloromethyl-8-methoxypsoralen (HMT), angelicin (isopsoralen), 5-methylangelicine (5 MIP), 3-carboxypsoralen, porphyrin, hematoporphyrin derivative (HPD), Photofrin II, benzoporphyrin derivative (BPD), protoporphyrin IX (PpIX), ether dye of hematoporphyrin (DHE), polyhematoporphyrin (PHE) esters, 13,17-N, N, N-dimethylethyl ethanolamine protoporphyrin ester (PH1008), tetra (3-hydroxyphenyl) -porphyrin (3-THPP), tetraphenylporphyrin monosulfonate (TPPS1) ), tetraphenylporphyrin disulfonate (TPPS2a), dihematoporphyrin ether, mesotetraphenylporphyrin, mesotetra (4N-methylpyridyl) porphyrin (T4MpyP), octa- (4-tert-butylphenyl) tetrapyrazineporphirazine (OPTP), phthalocyanine, tetra- (4-tert-butyl) ) phthalocyanine (t4-PcH2), tetra- (4-tert-t-butyl) phthalocyanatomagnesium (t4PcMg), sulfonated chloraluminium phthalocyanine (CASPc), chloraluminium phthalocyanine tetrasulfate (AIPcTS), mono-sulfonated aluminum phthalocyanine (AISPc) , di-sulfonated aluminum phthalocyanine (AlS2Pc), aluminum phthalocyanine tri-sulphonated nitride (AlS3Pc), tetra-sulfonated aluminum phthalocyanine (AlS4Pc), silicon phthalocyanine (SiPc IV), zinc phthalocyanine II (ZnPc), bis (diisobutyl-octadecylsiloxy) silicon 2, 3-naphthalocyanine (isoBOSINC), IV germanium octabutoxy-phthalocyanine (GePc), rhodamine 101 (Rh-101), rhodamine 110 (Rh-110), rhodamine 123 (Rh-123), rhodamine 19 (Rh-19), rhodamine 560 (Rh-560), rhodamine 575 (Rh-575), rhodamine 590 (Rh-590), rhodamine 610 (Rh-610), rhodamine 640 (Rh-640), rhodamine 6G (Rh-6G), rhodamine 700 (Rh-700), rhodamine 800 (Rh-800), rhodamine B (Rh-B), sulforhodamine 101, sulforhodamine 640, sulforhodamine B, coumarin 1, coumarin 2, coumarin 4, coumarin 6, coumarin 6H, coumarin 7, coumarin 30, coumarin 47, coumarin 102, coumarin 106, coumarin 120, coumarin 151, coumarin 152, coumarin 152A, coumarin 153, coumarin 311, coumarin 307, coumarin 314, coumarin 334, coumarin 337, coumarin 343, coumarin 440, coumarin 450, coumarin 456, coumarin 460, coumarin 461 , coumarin 466, cu marine 478, coumarin 480, coumarin 481, coumarin 485, coumarin 490, coumarin 500, coumarin 503, coumarin 504, coumarin 500, coumarin 510, coumarin 515, coumarin 519, coumarin 521, coumarin 522, coumarin 523, coumarin 535, coumarin 540, coumarin 540A , coumarin 548, 5-ethylamino-9-diethylaminbenzo [a] -phenoxazinium (EtNBA), 5-ethylamino-9-diethylaminobenzo [a] phenothiazinium (EtNBS), 5-ethylamino-9-diethylaminobenzo [a] phenenelenazinium (EtNBSe), chlorpromazine, chlorpromazine derivatives, chlorophyll derivatives, bacteriochlorophyll derivatives, metal ligand complexes, tris (2,2'-bipyridine) utenium (II) dichloride (RuBPY), tris (2,2'-bipyridine) dichloride rhodium (II) (RhBPY), tris (2,2'-bipyridine) dichloride platinum (II) (PthPY), pheophorbide a, merocyanin 540, vitamin D, 5-amino-levulinic acid, photosan, chlorin e6, ethylene diamine chlorin e6 , mono-L-aspartyl chlorin e6, derivatives of Nile blue of phenoxyzine, stilbene, stilbene derivatives 4- (N- (2-hydroxyethyl) -N-methyl) -aminophenyl) -4 '- (6-hydroxy-xylsulfonyl) -stilbene (APSS) and normal biological dyes and stains.

15. A method according to any of the preceding claims, further characterized in that the step of treating the particular volume of material with sufficient light to promote an excitation of the at least one photoactive molecular agent contained in the particular volume of the material includes the steps from: (al) modulate light coming from a light source with a modulation type, whereby a modulated light is produced; and (a2) treating the particular volume of the material with sufficient modulated light to promote a two-photon excitation of the at least one photoactive molecular agent in the particular volume of the material; and further includes the steps of: (d) demodulating the energy signal detected with the particular type of modulation; and (e) producing a demodulated energy signal that is characteristic of the particular volume of the material.

16. A method according to claim 15, further characterized in that the step of demodulating the energy signal detected with the type of modulation includes demodulating the energy signal detected at a frequency twice that of the particular type of modulation, whereby the second harmonic of the modulation type is detected.

A method according to claim 15 or claim 16, further characterized in that the demodulated energy signal that is characteristic of the particular volume of the material represents a change in longevity of at least one photoactivated molecular agent present in the particular volume of the material.

A method according to any of the preceding claims, further characterized in that the step of treating includes focusing a beam of light over a range of focal lengths such that a focal plane of the light beam extends to a location between a surface of the material and a point substantially beyond the material surface, whereby the treatment step can be extended to penetrate deep into the material.

19. A method according to claim 18, further characterized in that it also includes varying, while the light beam extends, the position of focal length within the material, so that the steps of photoactivation, detection and production of a signal of detected energy occur along positions between the material surface and a position located substantially beyond the material surface, whereby the imaging is three dimensional.

20. A method according to any of the preceding claims, further characterized in that the light comprises light near the infrared.

21. A method according to any of the preceding claims, further characterized in that the light is a focused beam of light.

22. A method according to any of the preceding claims, further characterized in that the light comprises laser light.

23. A method according to claim 22, further characterized in that the laser produces pulse energies of approximately 20 nanojoules.

A method according to any of the preceding claims, further characterized in that the step of treating includes operating a laser to produce a pulsed output having a pulse repetition frequency of greater than about 75 megahertz and a pulse duration less than nanoseconds

25. A method according to any of the preceding claims, further characterized in that the detection step comprises detecting emitted light that does not trace back to the origin of an optical path of the incident light coming from the light source.

26. A method according to any of the preceding claims, further characterized in that the light comprises laser light and wherein treating further includes modulating the light beam and wherein one of the detection step or the production steps includes using a stage of wavelength selection.

27. A method according to claim 26, further characterized in that the steps of treating and photoactivating are arranged to produce emitted light which comes from the molecular agent in the material and is essentially synchronous with a modulation of the laser light.

28. A method according to any of the preceding claims, for medical imaging, comprising the steps of: introducing a photoactive molecular agent into a tissue, the agent is selected for specificity of the tissue of interest, the agent being susceptible to two photon excitation (TPE); allow the agent to accumulate in a specific tissue of interest; orient light to specific regions of interest within the tissue, including regions substantially below a tissue surface, light is selected in frequency and energy to penetrate the tissue and promote TPE substantially only in the focal region; control the location of the confocal region over a range of depths within the tissue; use TPE, photoactivating the agent over the range of depths within the tissue, with which photoactivated agents are produced in the confocal region, where the photoactivated molecular agent emits energy; detect the emitted energy; and producing a detected energy signal that is characteristic of the tissue in the focal region.

29. A method according to claim 28, further characterized in that the step of orienting light includes generating light near infrared using a pulsed laser that operates at short pulse widths and a high pulse repetition rate, and focusing the laser on the tissue.

30. A method according to claim 28 or claim 29, further characterized in that the step of controlling the location comprises varying the position of the confocal region with respect to the tissue under examination or varying the position of the tissue under reactive examination to a region. fixed focal

31. A method according to any of the preceding claims, further characterized in that the photoactivated molecular agent emits fluorescent or phosphorescent radiation.

32. An apparatus for medical imaging, characterized in that it comprises: a light source means for orienting light to or in tissue to be imaged, the light is selected in frequency and energy to penetrate into or under a tissue surface and to promote excitation of two photons substantially only in one region to be imaged; and a detection means positioned to receive and detect isotropic radiation emitted by a photoactivated molecular agent within the tissue after the agent has been excited using two-photon excitation.

33. An apparatus according to claim 32, further characterized in that it further comprises means for varying a position of the region to be imaged by light within a range of depths in the tissue to be imaged.

34. An apparatus according to claim 32 or claim 33, further characterized in that the light source means includes a means for producing a beam of collimated light; and wherein the light source means includes a focusing means for focusing the beam of light collimated to a focal region located within the tissue at a point below the surface.

35. An apparatus according to any of claims 33 to 34, for forming in image a particular volume of plant or animal tissue containing at least one photoactive molecular agent, characterized in that the apparatus comprises: a source of collimated light, having light an effective frequency to penetrate substantially into tissue, light adapts to promote simultaneous excitation of two photons of the molecular agent contained within the tissue; and focusing the apparatus to focus the collimated light through a range of focal lengths extending from a tissue surface to a depth substantially beyond the surface, the light source and the focusing apparatus work together to promote excitation of two photons (TPE) of the molecular agent, wherein a focal point or focal plane is adjusted with respect to the tissue.

36. An apparatus according to claim 35, further characterized in that it further comprises a detector located close to the tissue and placed to detect the light emitted by the molecular agent and which travels along a path that does not date back to the origin of an optical path of the incident light in the tissue, the detector is configured to produce a detected signal characteristic of the particular volume in which the light source has been focused.

37. An apparatus according to any of claims 32 to 36, further characterized in that the light source produces light near the infrared.

38. An apparatus according to any of claims 32 to 37, further characterized in that the light source produces a pulsed output.

39. An apparatus according to claim 38, further characterized in that the pulse frequency is in the range from about 1 kilohertz to about 10 gigahertz.

40. An apparatus according to claim 39, further characterized in that the pulse repetition frequency is greater than about 75 megahertz.

41. An apparatus according to any of claims 38 to 40 further characterized in that the light source produces a pulse duration of less than nanoseconds.

42. An apparatus according to any of claims 38 to 41, further characterized in that the pulses have an energy from about 10 picojoules to about 50 milijoules.

43. An apparatus according to claim 42, further characterized in that the light source produces pulse energies of approximately 20 nanojoules.

44. An apparatus according to any of claims 32 to 43, further characterized in that the light source comprises a laser.

45. An apparatus according to any of claims 32 to 44, further characterized in that it further comprises a processor coupled to the detector.

46. An apparatus according to claim 45, further characterized in that it further comprises a modulation system associated with the light source, the processor is coupled to the modulation system.

47. An apparatus according to any of claims 32 to 46, further characterized in that the light is an unfocused beam of light.

48. An apparatus according to any of claims 32 to 46, further characterized in that the light is a focused beam of light.

49. An apparatus according to any of claims 32 to 48, further characterized in that the light source means is used to orient light in and within the deep tissue to be imaged.

50. An apparatus according to any of claims 32 to 49, further characterized in that the light is selected so as to promote excitation of two photons substantially only in a focal region.

51. An apparatus according to any of claims 32 to 34 and 36 to 50, further characterized in that the excitation of two photons is simultaneous excitation of two photons.

52. The use of a photoactive molecular agent for the manufacture of an effective agent to diagnose diseased or diseased disease.

53. The use according to claim 52, further characterized in that the diseased disease or tissue comprises a human cancer.

54. The use according to claim 52 or claim 53, further characterized in that the photoactive molecular agent is capable of simultaneous excitation of two photons.

55. The use according to any of claims 52 to 54, further characterized in that the photoactive molecular agent comprises an agent as defined in any of claims 10 to 14.